Pulsatillae Radix, the root of Pulsatilla chinensis (Bge.) Regel, is recorded in the Pharmacopoeia of the People's Republic of China and has been widely used for its pharmacological activities, such as anti‐inflammatory, antioxidant, antibacterial, antitumor, and cardiovascular benefits. However, there are several look‐alike species that can be marketed as Pulsatillae Radix. To distinguish P. chinensis (Bge.) Regel from its look‐alikes, viz. Pulsatilla cernua (Thunb.) Bercht et Opiz., Pulsatilla dahurica (Fisch.) Spreng., Anemone tomeutosa (Maxim.) Pei., and Rhaponticum uniflorum (L.) DC, we used ultra high performance liquid chromatography with time‐of‐flight mass spectrometry combined with principal component analysis to compare their chemical compositions. Four ions, a (RT 8.98 min, m/z 1381.6671), b (RT 10.64 min, m/z 1219.6143), c (RT 11.52 min, m/z 1217.5978), and d (RT 13.6 min, m/z 749.4463), from P. chinensis (Bge.) Regel were identified as potential chemical markers to distinguish it from look‐alike species using an unsupervised statistical model combined with orthogonal partial least‐squares discriminant analysis. The results of this study provide an effective method for identifying and distinguishing P. chinensis (Bge.) Regel from similar plants.